Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC, pcDNA-FEZF1-AS1, anti-miR-NC, anti-miR-363-3p, miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1, Ki67 and cleaved caspase-3. RESULTS Compared with control group, the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）, and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group, the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group, the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group, the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）, the apoptotic rate was significantly increased （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）, and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

Dasatinib inhibits the viability and migration of renal carcinoma cell lines 786-O and 769-P by up-regulating p53 expression and down-regulating AKT phosphorylation in vitro

AIM To explore the effect of dasatinib on the viability, apoptosis and migration of human renal carcinoma cell lines 786-O and 769-P, as well as the molecular mechanism in vitro. METHODS 786-O cells and 769-P cells were treated with different concentrations （0~2 μmol/L） of dasatinib, and 0 μmol/L dasatinib was used as blank control group. MTT method was used to detect cell viability. Wound healing assay was used to detect the effect of dasatinib on migration. Hoechst 33258 staining was used to observe the effect of dasatinib on apoptosis. Flow cytometry was used to detect the effect of dasatinib on cell cycle. Western blot method was used to detected cell cycle- and apoptosis-related protein levels. RESULTS Dasatinib inhibited viability and migration of 786-O and 769-P cells, and the inhibitory effect of dasatinib increased with the concentration of dasatinib （P<0.05）. The IC₅₀ values of dasatinib against 786-O and 769-P cell lines were （0.958 7±0.028 8） μmol/L and （0.784 3±0.066 0） μmol/L, respectively. After treatment with dasatinib for 24 h, the expression of pro-apoptotic proteins cleaved caspase-3 and cleaved caspase-9 increased significantly （P<0.01）, while the expression of cyclin D1 decreased （P<0.05）. The cycle-related pathway proteins p53 and p21 increased （P<0.05）, while the level of p-AKT was decreased （P<0.05）. CONCLUSION Dasatinib impaired the viability and migration ability of human renal carcinoma cell lines 786-O and 769-P by up-regulating p53 expression and down-regulating AKT phosphorylation.     

Resveratrol protects cortical neurons in infant rats with bacterial meningitis through miR-223-3p/NLRP3 pathway

AIM To investigate the protective effect of resveratrol (Res) on cortical neurons in rat bacterial meningitis (BM) model. METHODS Group B hemolytic Streptococcus was injected via the posterior cistern to establish a BM model. Resveratrol was administered intranasally and microRNA-223-3p (miR-223-3p) antagomir was administered by intracerebroventricular injection. HE staining was used to observe the pathological changes of the brain tissue. Loeffler scoring method was used to evaluate the neurobehavioral functions. TUNEL staining was used to detect neuronal apoptosis. The expression of interleukin-1β (IL-1β), IL-18, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba1) was detected by immunofluorescence staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3）, cleaved caspase-1, IL-1β and IL-18 were determined by Western blot. The expression level of miR-223-3p was detected by RT-qPCR. Online software TargetScan was used to search for the complementary nucleotide sequences between miR-223-3p and NLRP3 mRNA. RESULTS Compared with sham group, the thickness of meninges in BM model was increased, the neurological score was decreased (P<0.05), and the number of TUNEL positive neurons was increased significantly (P<0.05). Astrocytes and microglia were activated, the fluorescence intensity of IL-1β and IL-18 was increased (P<0.05), and the expression levels of NLRP3, cleaved caspase-1, IL-1β, IL-18 and miR-223-3p were increased (P<0.05). Compared with BM group, after treatment with resveratrol, the neurological score was increased (P<0.05), the number of TUNEL positive neurons was decreased significantly (P<0.05), and the inflammatory response of astrocytes and microglia was suppressed. The fluorescence intensity of IL-1β and IL-18 was decreased (P<0.05), the protein levels of NLRP3, cleaved caspase-1, IL-1β and IL-18 were decreased (P<0.05), and the expression level of miR-223-3p was increased (P<0.05). A nucleotide sequence in the 3'-UTR of NLRP3 mRNA might be targeted by miR-223-3p. In the brain of rat BM model, compared with antagomir control group, the expression of NLRP3 was increased in miR-223-3p antagomir group with resveratrol treatment (P<0.05). CONCLUSION Resveratrol may reduce the inflammatory death of cortical neurons in BM model of infant rats through miR-223-3p/NLRP3 pathway, thus playing a protective role for the neurons.     

Expression of histone chaperone ASF1B in prostate cancer cells and its effect on cell viability in vitro

AIM To investigate the expression of histone chaperone anti-silencing function 1B （ASF1B） in prostate cancer cells and its effect on cell viability in vitro. METHODS Human prostate cancer PC-3 cells were used, and knockdown of ASF1B was conducted by small interfering RNA （siRNA） transfection into the cells. The cells were divided into control group, siRNA negative control vector （mock） group and siRNA-ASF1B group. The viability of the PC-3 cells treated with ASF1B-siRNA for 12, 24 and 48 h was measured by MTT assay. Flow cytometry was used to analyze cell apoptosis and cell cycle distribution. The mRNA expression of apoptosis-related molecules was detected by RT-qPCR, and the expression levels of MAPK/JNK/ERK signaling pathway-related proteins were determined by Western blot. RESULTS The protein level of ASF1B in the normal cells （benign prostatic hyperplasia） was significantly lower than that in the PC-3 cells （P<0.01）. Compared with control group and mock group, the protein expression level of ASF1B in the PC-3 cells transfected with siRNA-ASF1B plasmid and the viability of the PC-3 cells were significantly decreased （P<0.01）. The apoptotic rate of the PC-3 cells transfected with siRNA-ASF1B plasmid was significantly higher than that in control group （P<0.01）, and the cell cycle was arrested at G₁ phase. The mRNA levels of p53, caspase-3, Bax and PARP-1 in the PC-3 cells transfected with siRNA-ASF1B plasmid were up-regulated compared with those in control group and Mack group （P<0.01）. In addition, the protein levels of MAP2K4 and p-JNK in the PC-3 cells in siRNA-ASF1B group were significantly higher than those in mock group （P<0.01）, while the protein level of p-ERK was significantly lower than that in mock group （P<0.01）. CONCLUSION ASF1B silencing induces G₁ arrest and promotes apoptosis of PC-3 cells. Activating MAPK/JNK/ERK signaling pathway may be a possible contributor to the anti-prostate cancer effect of siRNA-ASF1B.     

circRNA-42398 inhibits activation of hepatic stellate cells via TGF-?β1/Smads signaling pathway modulation

AIM To study the effect of mouse circular RNA-42398 (mmu_circ_42398) over-expression on the activation of hepatic stellate cells. METHODS Mouse hepatic stellate JS1 cells were cultured and randomly divided into control group, vector group and mmu_circ_42398 over-expression group.mmu_circ_42398 over-expression plasmid vector was constructed, and then transiently transfected into JS1 cells using Lipofectamine 2000. The cells were collected 48 h after transfection. Expression of mmu_circ_42398 was detected by RT-qPCR.The backsplice site of PCR products was verified by sequencing. The protein levels of α-smooth muscle actin (α-SMA), collagen type I （Col I), transforming growth factor β1(TGF-β1), Smad2, Smad3, p-Smad2 and p-Smad3 in the cells were determined by Western blot. RESULTS RT-qPCR results showed that the expression of mmu_circ_42398 was significantly increased after mmu_circ_42398 over-expression vector was transiently transfected into the JS1 cells (P<0.01). The protein expression levels of α-SMA and Col I were significantly decreased(P<0.01), and the phosphorylation levels of Smad2 and Smad3 were decreased significantly in mmu_circ_42398 over expression group (P<0.01). However, the protein expression levels of TGF-β1, Smad2 and Smad3 had no significant change (P>0.05). CONCLUSION mmu-circ-42398 inhibits the activation of hepatic stellate cells via TGF-β1/Smads signaling pathway modulation.     

Alleviating effect of exenatide on ectopic lipid accumulation in skeletal muscle of ob/ob mice is independent on reducing body weight

AIM To investigate the alleviating effect of exenatide （Exe）, a glucagon-like peptide-1 （GLP-1） receptor agonist, on the ectopic lipid accumulation in skeletal muscle of ob/ob mice and its mechanism. METHODS Eight-week-old male ob/ob mice and their wild-type （WT） littermates were randomly divided into 3 groups, ob/ob group, ob/ob+Exe group and WT group, and treated with Exe at 24 nmol/kg or the same volume of saline intraperitoneally once daily for 4 weeks. The body weight, fasting blood glucose （FBG） and fat content were measured after the 4-week treatment. The oil red O staining and the quantification of triglyceride （TG） were performed on the skeletal muscle. The serum levels of TG, total cholesterol and free fatty acid （FFA） were also measured by ELISA. The expression levels of AMP-activated protein kinase （AMPK） and lipid metabolism-related proteins were determined by Western blot. Mouse myoblast C2C12 cells were used as an in vitro model to further investigate the effects of Exe. RESULTS As compared with the ob/ob mice treated with saline, 4-week Exe treatment did not reduce body weight, FBG, food intake and fat content in ob/ob mice （P>0.05）. However, serum FFA was decreased （P<0.05）. Oil red O staining and the quantification of TG showed that 4-week Exe treatment significantly attenuated the ectopic lipid accumulation in the skeletal muscle of ob/ob mice （P<0.05）. The results of Western blot showed that the levels of phosphorylated AMPK （p-AMPK） and lipolysis-related proteins were up-regulated, while the lipid synthesis-related proteins were down-regulated by Exe （P<0.05）. Treatment with Exe alleviated the lipid accumulation in the C2C12 cells induced by sodium palmate （P<0.05）, and the effects of Exe on the levels of p-AMPK and lipid metabolism-related proteins in the C2C12 cells were consistent with those in the ob/ob mice （P<0.05）. Treatment with Exe also up-regulated the protein expression of glucose transporter 4 and improved the ability of glucose uptake in the C2C12 cells （P<0.05）. CONCLUSION Short-term Exe treatment attenuates the ectopic lipid accumulation in skeletal muscle of ob/ob mice by up-regulating lipolysis-related proteins and down-regulating lipid synthesis-related proteins, which is independent on body weight loss.     

Inhibitory effect of Wendan decoction on atherosclerotic plaque formation in ApoE-/- mice by regulating expression of membrane proteins involved in reverse cholesterol transport

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on reverse cholesterol transport. METHODS Eight-week-old apolipoprotein E gene knockout （ApoE^-/-） mice with high-fat diet and daily drug gavage were randomly divided into model group, simvastatin group, and low-, middle- and high-dose Wendan decoction groups, with 15 mice in each group. The C57BL/6 mice of the same age served as control group. The mice were weighed once every week. After 10 weeks, the mice were anesthetized with chloral hydrate. The serum were collected for lipid level examination. The atherosclerotic plaque buildup in aortic root and whole aorta was observed by HE staining and oil red O staining, respectively. The levels of proteins related to cholesterol transport, ATP-binding cassette transporter A1 （ABCA1） and caveolin-1 in the aorta, and scavenger receptor class B type I （SR-BI） and CD36 in the liver, were quantified by Western blot. RESULTS Wendan decoction at middle dose inhibited the increase in the body weight of ApoE^-/- mice fed with high-fat diet （P<0.05）. Wendan decoction at different doses significantly reduced the serum levels of triglyceride, total cholesterol and low-density lipoprotein cholesterol in the ApoE^-/- mice （P<0.05 or P<0.01）, but had no effect on serum high-density lipoprotein cholesterol level （P>0.05）. Wendan decoction at different doses inhibited the formation of atherosclerotic plaques in whole aorta of the ApoE^-/- mice （P<0.05 or P<0.01）. Middle- and high-dose Wendan decoction significantly inhibited the formation of atherosclerotic plaques in the aortic root （P<0.05）. Bedsides, Wendan decoction at different doses increased the protein level of ABCA1 and decreased the protein level of caveolin-1 in the aorta of the ApoE^-/- mice （P<0.01）. Middle- and high-dose Wendan decoction increased the liver protein level of SR-BI in the ApoE^-/- mice （P<0.01）. However, Wendan decoction at different doses had no effect on the liver protein level of CD36 in the ApoE^-/- mice （P>0.05）. CONCLUSION Wendan decoction reduces the body weight, serum lipid levels and formation of atherosclerotic plaques in ApoE^-/- mice fed with high-fat diet, and its mechanism is related to up-regulation of ABCA1 protein level in the aorta and SR-BI protein level in the liver as well as down-regulation of caveolin-1 protein level in the aorta.     

Curcumin inhibits Porphyromonas gingivalis LPS-induced inflammatory response in human gingival fibroblasts by up-regulating miR-124

AIM To investigate the effects of curcumin （Cur） on the inflammatory response of human gingival fibroblasts （HGFs） induced by Porphyromonas gingivalis （Pg） lipopolysaccharide （LPS） and the role of microRNA-124 （miR-124） in this process. METHODS The HGFs were divided into control group, LPS group （10 mg/L LPS） and LPS+Cur （20, 40 and 80 μmol/L） groups （10 mg/L LPS+corresponding dose of Cur）. After treatment for 24 h, CCK-8 assay was used to measure the cell viability. ELISA was used to measure the levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in the supernatant. The level of miR-124 in the cells was detected by RT-qPCR. The protein levels of nuclear factor kappa B （NF-κB） p-p65 in cytoplasm and nucleus were determined by Western blot, and the nuclear translocation of NF-κB p-p65 was evaluated by laser confocal microscopy. After transfection with mimic-NC or miR-124 mimic, the expression of miR-124 and NF-κB p-p65 protein in the cytoplasm and nucleus of the cells were also detected. RESULTS The cell viability, the level of miR-124 in the cells and NF-κB p-p65 protein level in cytoplasm of LPS group were lower than those in control group （P<0.05）, while the levels of IL-1β and TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were higher than those in control group （P<0.05）. The cell viability, the level of miR-124 in cells and NF-κB p-p65 protein level in the cytoplasm of LPS+Cur （40 and 80 μmol/L） groups were higher than those in LPS group （P<0.05）, while the level of TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were lower than those in LPS group （P<0.05）. The level of IL-1β in the supernatant of LPS+80 μmol/L Cur group was lower than that in LPS group （P<0.05）. The levels of miR-124 and NF-κB p-p65 protein level in the cytoplasm of miR-124 mimic group were higher than those in LPS group and mimic-NC group （P<0.05）, while the level of NF-κB p-p65 proteinlevel in the nucleus was lower than that in LPS group and mimic-NC group （P<0.05）. CONCLUSION Curcumin inhibits the inflammatory response of HGFs induced by Pg LPS, which may be achieved by up-regulating miR-124 and then inhibiting the nuclear translocation of NF-κB p-p65.     

Mangiferin attenuates hypoxia/reoxygenation-induced injury of human myocardial cells

AIM To investigate the effect of mangiferin on hypoxia/reoxygenation (H/R)-induced injury of human myocardial cells and its mechanism. METHODS Human myocardial AC16 cells were divided into normal group, H/R group and H/R + mangiferin (50, 100 and 200 μmol/L) treatment groups. The mRNA and protein expression levels of Kelch-like epichlorohydrin-associated protein-1 (Keap-1), Bax, Bcl-2, caspase-3, caspase-9 and superoxide dismutase 2 (SOD2) were detected by RT-qPCR and Western blot, respectively. The protein expression of nuclear factor E2-related factor 2 (Nrf-2) in nucleus was determined by Western blot. The expression of microRNA-432-3p (miR-432-3p) was detected by RT-qPCR. The generation of reactive oxygen speciess (ROS) in the cells was measured by DCFH-DA probing. The cell viability was measured by CCK-8 assay. Apoptosis was analyzed by flow cytometry. RESULTS No significant difference in the expression of miR-432-3p and Keap-1 between normal group and H/R group was observed. Compared with normal group, the nuclear translocation of Nrf-2, the ROS level, and the mRNA and protein expression of Bax, caspase-3 and caspase-9 were significantly increased in H/R group （P<0.05）. The mRNA and protein expression of SOD2 and Bcl-2, and the cell viability significantly decreased in H/R group compared with normal group, while the apoptosis was increased significantly （P<0.05）. Treatment with mangiferin resulted in an increase in the miR-432-3p expression and a decrease in the ROS level, and the expression of Keap-1, Bax, caspase-3 and caspase-9 was also inhibited as compared with H/R group （P<0.05）. The Nrf-2 nuclear translocation, and the protein levels of SOD2 and Bcl-2 in mangiferin treatment groups were significantly increased as compared with H/R group （P<0.05）. The cell viability was increased significantly, and the apoptosis was decreased significantly in mangiferin treatment groups as compared with H/R group （P<0.05）. The effects of mangiferin in middle- and high-dose groups were better than those in low-dose group, and no significant difference between middle- and high-dose groups was found. CONCLUSION Mangiferin inhibits the decrease in myocardial cell viability and the apoptosis induced by H/R injury. The mechanism may be related to the up-regulation of Nrf-2 antioxidant stress effect via enhancing the expression of miR-432-3p.     

Inhibitory effect of Wendan decoction on formation of foam cells induced by ox-LDL

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on formation of foam cells. METHODS The optimal concentrations of Wendan decoction without cytotoxity to cells were selected by MTT assay. After Wendan decoction treatment, the formation of foam cells was examined by oil red O staining. The cholesterol efflux, cholesterol level, free cholesterol level and cholesterol esterification rate were analyzed using cholesterol efflux assay, total cholesterol assay and free cholesterol assay. The expression levels of macrophage membrane proteins, including CD36, scavenger receptor class A （SR-A）, ATP-binding cassette transporter A1 （ABCA1） and scavenger receptor class B type I （SR-BI）, were quantified by Western blot. RESULTS The optimal concentrations of Wendan decoction without cytotoxity to the cells were 0~6 g/L. Wendan decoction at the concentrations of 1.5, 3 and 6 g/L were selected for the experiments. Wendan decoction at these concentrations inhibited the formation of foam cells induced by oxidized low-density lipoprotein （ox-LDL）, and reduced the accumulation of intracellular lipids in a concentration-dependent manner （P<0.05 or P<0.01）. Wendan decoction also reduced intracellular total cholesterol level, cholesterol ester level and cholesterol esterification rate （P<0.05 or P<0.01）, promoted efflux of intracellular cholesterol （P<0.01）, and decreased the protein level of CD36 in THP-1 cell-derived macrophages （P<0.01） in a concentration-dependent manner. Wendan decoction at the concentration of 6 g/L significantly reduced the protein level of SR-A in THP-1 cell-derived macrophages （P<0.05）. At the concentrations of 3 and 6 g/L, Wendan decoction significantly increased the protein levels of ABCA1 and SR-BI in THP-1 cell-derived macrophages （P<0.05 or P<0.01）. CONCLUSION Wendan decoction significantly inhibits ox-LDL-induced formation of foam cells by reducing cholesterol deposition and promoting cholesterol efflux, and its mechanism may be related to the down-regulation of CD36 and SR-A and the up-regulation of ABCA1 and SR-BI.     

Effect of abnormal expression of PDK4 on glycolysis and proliferation in prostate cancer cells

AIM To investigate the expression of pyruvate dehydrogenase kinase 4 （PDK4） in prostate cancer tissue and its effect on glycolysis and growth of prostate cancer cells. METHODS Immunohistochemistry was used to compare the expression differences of PDK4 protein in benign prostatic hyperplasia （BPH） and prostate cancer tissues. The expression levels of PDK4 in normal prostatic epithelial cells （RWPE-1） and different prostate cancer cell lines （PC3, LNCaP, DU145 and C4-2） were detected by RT-qPCR and Western blot. Recombinant plasmid carrying PDK4-shRNA was constructed, and the expression of PDK4 in prostate cancer PC3 cells was down-regulated by transfection with PDK4-shRNA. The changes in glycolysis level of PC3 cells before and after transfection were determined by cell glycolysis kit, and the effects of PDK4 on the viability and cell cycle distribution of PC3 cells were detected by CCK-8 assay and flow cytometry. RESULTS In prostate cancer tissues, the expression level of PDK4 protein was significantly higher than that in BPH tissues （P<0.05）, and the analysis of immunohistochemical score showed that prostate cancer tissues with high Gleason score displayed significantly higher PDK4 expression than those with low Gleason score （P<0.05）. Compared with normal prostatic epithelial cells, RT-qPCR and Western blot results indicated that the expression level of PDK4 was also significantly increased in prostate cancer cell lines （P<0.05）. In addition, CCK-8 assay results showed that the viability of prostate cancer PC3 cells was significantly decreased after knockdown of PDK4 expression （P<0.05）. The results of flow cytometry demonstrated that knockdown of PDK4 expression in PC3 cells resulted in a notable increase in G₀/G₁ phase arrest （P<0.05）. CONCLUSION PDK4 is highly expressed in prostate cancer tissues and cell lines, and significantly increases in prostate cancer with high Gleason score. In addition, down-regulation of PDK4 expression significantly inhibits glycolysis and growth of prostate cancer cells, resulting in cell cycle arrest at G₀/G₁ phase.     

Cyanidin attenuates pressure overload-induced cardiac remodeling in mice via mitochondrial fusion enhancement

AIM To investigate the effect of cyanidin （Cyn） on pressure overload-induced cardiac remodeling and the underlying mechanism. METHODS Six-week-old male C57BL/6 mice （n=120） were divided into 4 groups： sham group （n=20）, sham+Cyn group （n=20）, transverse aortic constriction （TAC） group （n=40） and TAC+Cyn group （n=40）. The model of cardiac chronic pressure overload was induced by TAC, and the first day of TAC was defined as day 0. The animals in sham+Cyn group and TAC+Cyn group were treated with Cyn dissolved in DMSO and normal saline （5 mg·kg^-1·d^-1） for 5 d before TAC, while the animals in sham group and TAC group were treated with the same amount of DMSO and normal saline. Four weeks after TAC, the survival rate of the animals in each group was analyzed, the heart function of the mice was measured by ultrasound echocardiography, and the heart weight/body weight and lung weight/body weight were calculated. The cross-sectional area of the cardiomyocytes was measured by wheat germ agglutinin staining and hematoxylin-eosin staining. The degree of cardiac oxidative stress was evaluated by dihydroethidium staining and measurement of superoxide dismutase （SOD） and malondialdehyde （MDA） levels. The cardiomyocyte apoptosis was detected by TUNEL method. The mRNA expression levels of atrial natriuretic peptide （ANP）, brain natriuretic peptide （BNP） and β-myosin heavy chain （β-MHC） were detected by RT-qPCR, and the protein expression levels of Bax, Bcl-2, optic atrophy protein 1 （OPA1） and dynamin-related protein 1 （Drp1） were determined by Western blot. The mitochondrial morphological changes were observed by transmission electron microscopy. RESULTS Compared with TAC group, the survival rate of the mice in TAC+Cyn group was significantly increased （P<0.05）, the myocardial apoptosis, the cross-sectional area of myocardial cells, the heart weight/body weight, the lung weight/body weight, the level of reactive oxygen species and the MDA content were decreased （P<0.05）, and the SOD was activated （P<0.05）. M-mode ultrasound tests showed that Cyn treatment significantly increased left ventricular ejection fraction and left ventricular fractional shortening in the mice after TAC （P<0.05）, while left ventricular end-diastolic diameter and left ventricular posterior wall thickness in diastole were reduced （P<0.05）. Transmission electron microscopic observation showed that the number of myocardial mitochondria was increased and the mitochondrial area was decreased after TAC （P<0.05）, while treatment with Cyn increased the area of myocardial mitochondria and decreased the mitochondrial number （P<0.05）. Compared with sham group, the protein level of OPA1 in TAC group was significantly reduced （P<0.05）, while treatment with Cyn significantly increased the protein level of OPA1. CONCLUSION Cyanidin significantly increases the survival rate, improves the cardiac function and attenuates the cardiac remodeling of the mice after TAC. The mechanism may be related to the inhibition of myocardial mitochondrial OPA1 cleavage and the promotion of mitochondrial fusion.     

Scutellarin affects LPS-induced oxidative stress and apoptosis of glomerular epithelial cells by up-regulating miR-7-5p

AIM To investigate the effect of scutellarin （SCU） on oxidative stress and apoptosis induced by lipopolysaccharide （LPS） in human glomerular epithelial cells and its mechanism. METHODS Human glomerular epithelial cells were cultured in vitro, and were treated with LPS （1.0 mg/L） to establish a cell injury model. The cells were divided into normal control （NC） group, LPS group, NC+SCU group, LPS+SCU group, LPS+miR-NC group, LPS+microRNA-7-5p （miR-7-5p） group, LPS+SCU+anti-miR-NC group and LPS+SCU+anti-miR-7-5p group. Cell viability was detected by CCK-8 assay. Apoptosis was detected by flow cytometry. The intracellular malondialdehyde （MDA） content and superoxide dismutase （SOD） activity, and lactate dehydrogenase （LDH） activity in the cell culture supernatant were determined by kit. RT-qPCR was used to detect the expression level of miR-7-5p. RESULTS Compared with NC group, the cell viability, miR-7-5p expression and SOD activity in LPS group were significantly reduced, and the apoptotic rate, MDA content and LDH activity were significantly increased （P<0.05）. Compared with LPS group, the cell viability, miR-7-5p expression and SOD activity in LPS+SCU group were significantly increased, and the apoptotic rate, MDA content and LDH activity were significantly reduced （P<0.05）. Compared with LPS+miR-NC group, the cell viability and SOD activity in LPS+miR-7-5p group were significantly increased, and the apoptotic rate, MDA content and LDH activity were significantly reduced （P<0.05）. Compared with LPS+SCU+anti-miR-NC group, the cell viability and SOD activity in LPS+SCU+anti-miR-7-5p group were significantly reduced, and the apoptotic rate, MDA content and LDH activity were significantly increased （P<0.05）. CONCLUSION Scutellarin inhibits LPS-induced oxidative stress damage and apoptosis in glomerular epithelial cells via up-regulating miR-7-5p expression.     

IL-33-modified BMSCs attenuate acute kidney injury induced by sepsis in rats through MyD88

AIM To investigate the effect of interleukin-33 (IL-33)-modified bone marrow mesenchymal stem cells (BMSCs) on sepsis-induced acute kidney injury (AKI) in rats and the expression of myeloid differentiation factor 88(MyD88). METHODS A septic rat model was established by cecal ligation and puncture. The SD rats (n=80) were randomly divided into control group, model group, negative transfection group (transplanting untransfected BMSCs) and IL-33 transfection group (transplanting BMSCs transfected with IL-33), with 20 in each group. Survival rates of the rats within 72 h in the 4 groups were compared. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured before, and 24, 48 and 72 h after transplantation. The kidney pathological damage was observed by HE staining, and the apoptosis of renal cells was detected by TUNEL method 72 h after transplantation. Western blot was used to detect the protein expression levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), Toll-like receptor 4 (TLR4) and MyD88. RESULTS The survival rate of the rats in model group was significantly lower than that in control group (P<0.05). The survival rate of the rats in IL-33 transfection group was higher than that in model group and negative transfection group (P<0.05). The levels of SCr and BUN in model group were higher than those in control group (P<0.05). The levels of SCr and BUN in IL-33 transfection group were significantly reduced after transplantation, and were lower than those in model group and negative transfection group (P<0.05). The renal tissue pathological injury score in model group was significantly higher than that in control group (P<0.05). Compared with model group and negative transfection group, the renal tissue pathological injury score in IL-33 transfection group was significantly reduced (P<0.05). The proportion of apoptotic cells in the kidney tissues in model group were higher than that in control group (P<0.05). Compared with model group and negative transfection group, the proportion of apoptotic cells in the kidney tissues in IL-33 transfection group was significantly reduced (P<0.05). The protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in model group were significantly higher than those in control group (P<0.05). Compared with model group and negative transfection group, the protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in IL-33 transfection group were significantly decreased (P<0.05). CONCLUSION IL-33 gene-modified BMSCs significantly improve the renal function of AKI rats with sepsis. The mechanism may be related to IL-33 regulating TLR4/MyD88 signaling pathway and inhibiting renal inflammatory response.     

miR-556-3p regulates viability,migration and invasion of endometrial cancer cells by targeting SASH1

AIM To investigate whether microRNA-556-3p （miR-556-3p） regulates the viability, migration and invasion of endometrial cancer cells by targeting SASH1 gene. METHODS The expression of miR-556-3p, and the mRNA and protein levels of SASH1 in endometrial cancer tissues were detected by RT-qPCR and Western blot. Anti-miR-556-3p or pcDNA-SASH1 was transfected into endometrial cancer Ishikawa cells. The cell viability was detected by MTT assay, the migration and invasion abilities of the cells were detected by Transwell chamber method, and the protein expression levels of cyclin D1, p21, matrix metalloproteinase-2 （MMP-2） and MMP-9 were detected by Western blot. StarBase prediction and dual-luciferase reporter experiments were used to analyze the targeting relationship between miR-556-3p and SASH1. Anti-miR-556-3p and si-SASH1 were co-transfected into the Ishikawa cells, and their effects on cell viability, migration and invasion were examined by the methods described above. RESULTS Compared with adjacent tissues, the expression of miR-556-3p in endometrial cancer tissues was increased significantly, and the expression of SASH1 at mRNA and protein levels was decreased significantly （P<0.05）. Inhibition of miR-556-3p expression or induction of SASH1 over-expression obviously reduced the viability of Ishikawa cells, the number of migratory cells, the number of invasive cells and the protein levels of cyclin D1, MMP-2 and MMP-9, and dramatically increased the protein level of p21 （P<0.05）. miR-556-3p targeted SASH1 and negatively regulated its expression. Knock-down of SASH1 expression reversed the inhibitory effect of miR-556-3p expression inhibition on the viability, migration and invasion of Ishikawa cells. CONCLUSION Inhibition of miR-556-3p expression suppresses the viability, migration and invasion of endometrial cancer cells. The mechanism is related to the regulation of its target gene SASH1.     

Aβ1-40 induces inflammation and phenotypic switching via activation of MAPKs signaling pathway in vascular smooth muscle cells

AIM To investigate the effects of amyolid β-protein 1-40 （Aβ1-40） on inflammation, viability, migration and phenotypic switching in vascular smooth muscle cells （VSMCs）, and to analyze the underlying mechanisms. METHODS The VSMCs were treated with Aβ1-40 at different concentration gradients for appropriate time. CCK-8 and Transwell assays were performed to evaluate the viability and migration ability of VSMCs. The levels of inflammatory factors including interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α）, phenotypic switching-related proteins including α?smooth muscle actin （α?SMA）, osteopontin （OPN） and Krüppel?like factor 4 （KLF4）, and mitogen-activated protein kinases （MAPKs） signaling pathway-related proteins including p-p38 MAPK, p-ERK1/2 and p-JNK were determined by Western bolt. RESULTS After Aβ1-40 treatment, the levels of inflammatory factors IL-1β and TNF-α in the VSMCs were significantly increased （P<0.05）, and the expression of phenotypic switching-related proteins was altered, as indicated by down-regulation of α?SMA and up-regulation of OPN and KLF4 （P<0.05）. Treatment with Aβ1-40 within a certain concentration range promoted the viability and migration of the VSMCs. In addition, the protein levels of p-p38 MAPK, p-ERK1/2 and p-JNK were significantly increased by Aβ1-40 treatment （P<0.05）. Furthermore, pretreatment with specific inhibitors of MAPKs pathway significantly reduced the levels of IL-1β and TNF-α （P<0.05）, and inhibited the phenotypic switching, as indicated by up-regulation of α?SMA and down-regulation of OPN and KLF4 （P<0.05）. CONCLUSION Treatment with Aβ1-40 induces the inflammation and phenotypic switching in VSMCs via activation of MAPKs signaling pathway.